Identification and characterization of antibodies aggregates or other intermolecular products of antibodies is of major importance as numerous antibodies are used as pharmaceutical products. The analysis of antibodies aggregates or aggregates formed by other therapeutic proteins as a consequence of aging, heat stress, pH stress or host cell expression is of crucial importance to evaluate the stability of these therapeutic molecules.
Aggregation of therapeutic proteins such as antibodies can become a major problem when application to a patient requires a high concentration to achieve therapeutic efficacy. The aggregation of antibodies and other therapeutic proteins has important consequences including decreased activity and solubility. A major consequence of therapeutic protein aggregation is also an increasing immunogenicity that can have consequences for the patients treated.
The aggregation of monoclonal antibodies and other therapeutic proteins is a complex problem to be addressed as the aggregation can affect the proteins differently depending on the type of stress involved. During production, monoclonal antibodies and other therapeutic proteins are submitted to temperature, pressure and pH variations that can lead to aggregations. The analysis of antibodies aggregation is of high interest as this phenomenon has an influence on the binding of these therapeutics on their targets (antigen, receptor, or the like).
Conventional technologies for the analysis of such antibodies aggregates include size exclusion chromatography (SEC), whereby the protein extract containing the antibody aggregate is separated from the monomeric form and collected in liquid phase; near- and far UV circular dichroism analysis, whereby the tertiary structure of monomers and dimers can be evaluated; capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), whereby antibodies aggregates can be characterized distinguishing between covalent and non-covalent aggregation. Using electrospray ionization (ESI) mass spectrometry, it is possible to distinguish between different species of antibody aggregates pre-fractionated by size exclusion chromatography combined with reverse phase chromatography (SEC/rp-HPLC) (Remmele, R. et al., J. Pharma. Sci. 95:126-145 (2005)). This ESI mass spectrometric analysis does not allow a quantitative analysis of the amount of aggregation of a sample as this analysis has to be applied to highly purified fractions of the aggregated sample.
MALDI mass spectrometry has been used for the analysis of intact non-covalent protein complexes. Many particular methodologies including specific matrix and laser combinations, accumulation of “first shot” spectra, modification of pH, selected solvent conditions during sample preparation, and use of crosslinking reagents to stabilize covalently the subunits of a complex have been developed. These procedures give interesting results but also have strong limitations (Farmer et al., Biological Mass Spectrometry 20:796-800, 1991; Farmer et al., J. Mass Spectrom. 33:697-704, 1998). Among these limitations, the low efficiency of the crosslinking stabilization protocol used and the impossibility to detect the intact protein complexes in the high-molecular range (higher than 200 kDa) are particularly noteworthy.
Recently, using the combination of specially developed MALDI high-mass detection and a new crosslinking protocol, Nazabal et al. were able to analyze protein-protein interactions with a higher level of complexity and a improved sensitivity in a high-mass range (Nazabal, A. et al., Anal. Chem. 78:3562-3570, 2006; WO 2006/116893).
The mentioned analytical methods have never been applied to the quantitative analysis of antibody or other therapeutic protein aggregates because MALDI mass spectrometry is cumulating three major disadvantages: 1) the laser used for the ionization disrupts the non-covalent interactions of the aggregates; 2) detection sensitivity is reduced or inexisting in the high-mass range; and 3) MALDI mass spectrometry is not considered a quantitative tool. Only one study by Farmer et al. (J. Mass Spectrom. 30:1245-1254, 1995) proposed a basis for the quantitative analysis of crosslinked oligomeric complexes using MALDI mass spectrometry. The method proposed the use of a correction factor to compensate the use of standard detection in the experiments. The use of standard detection systems leads to detection limitations in the higher mass range; limitations that may be compensated by a correction factor calculated for the 30-150 kDa range. This correction factor for detection efficiency is only applicable when using the same type of instrument and for exactly the same experimental set-up, extremely limiting this method for the quantitative analysis and making it difficult to apply.
In WO 2006/116893 (Eidgenössische Technische Hochschule Zürich) a mass spectrometric method is proposed wherein intact ions of undigested, unfragmented covalently stabilized supramolecular target-ligand complexes are analyzed with matrix assisted laser desorption ionization (MALDI). The method is illustrated by the analysis of antibody-antigen complexes and other protein-protein complexes such as complexes between CDC42 and Salmonella outer protein E, but has not been used for the analysis of self-aggregation of an antibody or a therapeutic protein. In addition, this method has never been used for quantitative analysis of the amount of protein complex formed.